US10335445B2 - Compositions and methods of antiallergic phorbol ester and phorbol derivatives as the main active ingredients from the seeds of aquilaria malaccensis - Google Patents
Compositions and methods of antiallergic phorbol ester and phorbol derivatives as the main active ingredients from the seeds of aquilaria malaccensis Download PDFInfo
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- IYLXPPIKNQIPEK-JUSIOJTQSA-N [H][C@@]12C=C(C)C(=O)[C@@]1(O)CC(CO)=C[C@]1([H])[C@]2(O)[C@H](C)[C@@H](OC(=O)/C=C\C=C\C=C\CCCCCCC)[C@@]2(OC(C)=O)C(C)(C)[C@]21[H] Chemical compound [H][C@@]12C=C(C)C(=O)[C@@]1(O)CC(CO)=C[C@]1([H])[C@]2(O)[C@H](C)[C@@H](OC(=O)/C=C\C=C\C=C\CCCCCCC)[C@@]2(OC(C)=O)C(C)(C)[C@]21[H] IYLXPPIKNQIPEK-JUSIOJTQSA-N 0.000 description 4
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- JHLLKSUTRIJADF-ZOHNXYPESA-N CCC=CCCCCCCCC.[H][C@@]12C=C(C)C(=O)[C@@]1(O)CC(COC(=O)CCCCCCC)=C[C@]1([H])[C@]2(O)[C@H](C)C[C@@]2(OC(C)=O)C(C)(C)[C@]21[H] Chemical compound CCC=CCCCCCCCC.[H][C@@]12C=C(C)C(=O)[C@@]1(O)CC(COC(=O)CCCCCCC)=C[C@]1([H])[C@]2(O)[C@H](C)C[C@@]2(OC(C)=O)C(C)(C)[C@]21[H] JHLLKSUTRIJADF-ZOHNXYPESA-N 0.000 description 1
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- YATZXAYMOLFCLR-FWEOTKLJSA-N [H][C@@]12C=C(C)C(=O)[C@@]1(O)CC(CO)=C[C@]1([H])[C@]2(O)[C@H](C)[C@@H](OC(=O)/C=C/C=C/C(O)C(O)CCCCCCC)[C@@]2(OC(C)=O)C(C)(C)[C@]21[H].[H][C@@]12C=C(C)C(=O)[C@@]1(O)CC(CO)=C[C@]1([H])[C@]2(O)[C@H](C)[C@@H](OC(=O)/C=C/C=C/C=O)[C@@]2(OC(C)=O)C(C)(C)[C@]21[H] Chemical compound [H][C@@]12C=C(C)C(=O)[C@@]1(O)CC(CO)=C[C@]1([H])[C@]2(O)[C@H](C)[C@@H](OC(=O)/C=C/C=C/C(O)C(O)CCCCCCC)[C@@]2(OC(C)=O)C(C)(C)[C@]21[H].[H][C@@]12C=C(C)C(=O)[C@@]1(O)CC(CO)=C[C@]1([H])[C@]2(O)[C@H](C)[C@@H](OC(=O)/C=C/C=C/C=O)[C@@]2(OC(C)=O)C(C)(C)[C@]21[H] YATZXAYMOLFCLR-FWEOTKLJSA-N 0.000 description 1
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- A61K36/18—Magnoliophyta (angiosperms)
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- A61K36/83—Thymelaeaceae (Mezereum family), e.g. leatherwood or false ohelo
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- A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
- A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
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- A61K31/23—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms
- A61K31/232—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms having three or more double bonds, e.g. etretinate
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- C07C67/56—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
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- C07C69/533—Monocarboxylic acid esters having only one carbon-to-carbon double bond
- C07C69/58—Esters of straight chain acids with eighteen carbon atoms in the acid moiety
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- C07C69/587—Monocarboxylic acid esters having at least two carbon-to-carbon double bonds
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- C07C69/73—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of unsaturated acids
- C07C69/732—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of unsaturated acids of unsaturated hydroxy carboxylic acids
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- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- C07C2603/40—Ortho- or ortho- and peri-condensed systems containing four condensed rings
Definitions
- compositions and methods of antiallergic phorbol ester and phorbol derivatives as the main active ingredients from the seeds of Aquilaria malaccensis are provided.
- allergic diseases are the hypersensitive immuno-response induced by specific foods or a number of factors in the environment that usually arouse some indispositions, such as allergic rhinitis, urticaria, atopic dermatitis, asthma, and even severe anaphylaxis.
- the symptoms involve red eyes, itchy rashes, rhinorrhea, dyspnea, and swelling. Therefore, some foods and the factors in the environment are so-called allergens.
- the environment is getting polluting by the growth of industry development, and the green lands and forests such as the Brazilian rainforest are substantively lost by the land development.
- the phenomenon in number increase and age decline of patients with allergic diseases is observed and arisen from the changed climate and environment due to above reasons.
- IgE-mediated allergy is a common immune system disorder, and the combinations with allergens release inflammatory chemicals such as histamine.
- Current treatments for allergies include the avoidances of known allergens and the usages of corticosteroids and antihistamines; additionally, the intravenous injection of epinephrine would be applied for suppressions of hypersensitive responses when severe allergies occurred.
- mast cells and their degranulation play a crucial role in IgE-mediated allergic inflammatory responses such as allergic rhinitis, acute asthma, and atopic eczema (Lian et al., Int. J. Mol. Sci., 16, 2252-2268, 2015).
- Beta-hexosaminidase is an enzyme released along with histamine from mast cells (RBL-2H3 cells) upon activation and serves as a well-accepted in vitro model in allergy (Dearman et al., Toxicology, 206, 195-205, 2005). Although today we are able to treat the symptoms of allergy, available medications have undesirable effects, especially within a prolonged use. Therefore, there is a need to search for alternative treatment, and natural sources are often considered as safe and easily available.
- Agarwood is a priceless fragrant resinous wood from Aquilaria species (Thymelaeaceae), which is formed as a defense mechanism to fend off pathogens.
- Agarwood is widely applied in traditional medicine preparations for cardiotonic, carminative, antiasthmatic, aphrodisiac, astringent remedy.
- Aquillaria agarwood has been found effective against diarrhea, dysentery, gout, rheumatism, paralysis and parasites, and it has been beneficial for skin diseases (Talukdar et al, Int. J. Pharm. Pharm. Sci., 6, 629-631, 2014).
- Aquilaria species was previously found to possess antidepressant (Yang et al., J.
- the alcoholic extract of A. malaccensis stein and bark exhibited cardiotonic activity (Pant et al., Phytochemistry, 19, 1869-1870, 1980), and cytotoxicity against Eagle's carcinoma of the nasopharynx and P-388 lymphocytic leukemia cells in vitro (Gunasekera et al., J. Nat. Prod., 44, 569-572, 1981).
- the aqueous extract showed antitrypanosomal (Dyary et al., Trop. Biomed., 31, 89-96, 2014), antibacterial (Dash et al., Afr. J.
- Aquilaria malaccensis it is too slow growth of Aquilaria malaccensis to supply the stein and bark extracts plentifully and permanently. Relative to the stems and barks of agarwood ( A. malaccensis ), the seeds of A. malaccensis were easier to be obtained and thus chosen for alternative investigation to prevent and treat allergic diseases.
- the preparation methods, the compositions, and the bioactive assays including antiallergic, antiinflammatory, and cytotoxic tests of A. malaccensis seeds (AMS) extract, its fractions, and those isolated phorbol esters with formulas I ⁇ IV were designed and achieved.
- the primary object of the present invention is to provide antiallergic compositions from the seeds of Aquilaria malaccensis and their preparation methods.
- the second object of the present invention is to provide compositions from the seeds of Aquilaria malaccensis to prevent and treat the allergies and related hypersensitive immuno-responses effectively.
- the third object of the present invention is to provide compositions from the seeds of Aquilaria malaccensis to prevent and treat the allergies and related hypersensitive immuno-responses effectively, of which compositions comprise a compound represented by Formula I to VI and their isomers or a or a pharmaceutically acceptable salt thereof as an active ingredient.
- FIG. 1 shows preparation scheme of the present invention.
- FIG. 2 shows isolation scheme of AM4-3 to afford 15 subfractions.
- FIG. 3 shows isolation scheme of AM4-4 to afford 12 subfractions.
- FIG. 4 shows 1 H NMR spectra comparison of selected compositions from Aquilaria malaccensis seeds, the ethanolic extract (EtOH), 90% aqueous methanol layer (MeOH), AM-4, and AM4-4.
- FIG. 5 shows 1 H NMR spectrum of compound represented by Formula I.
- FIG. 6 shows 13 C NMR spectrum of compound represented by Formula I.
- FIG. 7 shows 1 H NMR spectrum of compound represented by Formula II.
- FIG. 8 shows 13 C NMR spectrum of compound represented by Formula II.
- FIG. 9 shows 1 H NMR spectrum of compound represented by Formula III.
- FIG. 10 shows 13 C NMR spectrum of compound represented by Formula III.
- FIG. 11 shows 1 H NMR spectrum of compound represented by Formula IV.
- FIG. 12 shows 13 C NMR spectrum of compound represented by Formula IV.
- FIG. 13 shows 1 H NMR spectrum of compound represented by Formula V.
- FIG. 14 shows 13 C NMR spectrum of compound represented by Formula V.
- FIG. 15 shows 1 H NMR spectrum of compound represented by Formula VI.
- FIG. 16 shows antiallergic activities of the compositions from Aquilaria malaccensis seeds by inhibition of ⁇ -hexosaminidase release.
- FIG. 17 shows Antiinflammatory effects of the compositions from Aquilaria malaccensis seeds on superoxide anion generation and elastase release in fMLP/CB-induced human neutrophils.
- FIG. 18 shows Cytotoxic activities of the compositions from Aquilaria malaccensis seeds against HepG2, MDA-MB231, and A549 carcinoma cell lines.
- FIG. 19 shows Activity of phorbol ester-rich fraction (AM4) and phorbol ester I on stimulant-free degranulation in RBL-2H3 cells.
- the present invention is further illustrated by the preparation schemes ( FIG. 1 ⁇ 3 ), the NMR spectra of phorbol esters represented by Formulas I to VI ( FIG. 4 ⁇ 15 ), and the bioactive results (Tab. 1 ⁇ 4), and the skills and methods used herein are described in detail:
- AM4-3-1 3.4 mg; AM4-3-2, 25.3 mg; AM4-3-3, 345.6 mg; AM4-3-4, 49.2 mg; AM4-3-5, 16.4 mg; AM4-3-6, 39.5 mg; AM4-3-7, 8.4 mg; AM4-3-8, 3.8 mg; AM4-3-9, 42.7 mg; AM4-3-10, 23.0 mg; AM4-3-11, 42.5 mg; AM4-3-12, 43.5 mg; AM4-3-13, 23.5 mg; AM4-3-14, 7.9 mg; and AM4-3-15, 38.3 mg) Of which subfractions, AM4-3-6 and AM4-3-13 are two phorbol ester-rich fractions.
- AM4-4-1 2.2 mg; AM4-4-2, 16.3 mg; AM4-4-3, 3.5 mg; AM4-4-4, 5.7 mg; AM4-4-5, 7.7 mg; AM4-4-6, 11.5 mg; AM4-4-7, 37.6 mg; AM4-4-8, 6.8 mg; AM4-4-9, 43.9 mg; AM4-4-10, 3.3 mg; AM4-4-11, 3.5 mg; and AM4-4-12, 23.5 mg) Of which subfractions, AM4-4-3, AM4-4-7, AM4-4-8 and AM4-4-9 are four phorbol ester-rich fractions.
- phorbol ester I is 12-O-(2Z,4E,6E)-tetradeca-2,4,6-trienoylphorbol-13-acetate with a molecular formula of C 36 H 50 O 8 deduced from HRESIMS data m/z 633.33980 [M+Na] + (calcd for C 36 H 50 O 8 Na, 633.33979); [ ⁇ ] D 25 : ⁇ 3.75 ⁇ 1.97 (c 0.0667, CHCl 3 ); UV (MeOH) ⁇ max (log ⁇ ): 303 (2.78), 233 (2.75) nm; and IR (neat) ⁇ max : 3413, 2965, 2922, 1710, 1615, 1377, 1258, 1092, 802 cm ⁇ 1 .
- the 1 H and 13 C NMR spectra of phorbol ester I are shown in FIG. 5 and FIG. 6 , respectively.
- phorbol ester II is 12-deoxy-13-O-acetoylphorbol-20-octadec-9-enoate with a molecular formula of C 40 H 62 O 7 deduced from HRESIMS data m/z 677.43884 [M+Na] + (calcd for C 40 H 62 O 7 Na, 677.43878); [ ⁇ ] D 25 : +2.91 ⁇ 0.49 (c 0.3333, CHCl 3 ); UV (MeOH) ⁇ max (log ⁇ ): 285 (2.78), 250 (2.83) nm; and IR (neat) ⁇ max : 3409, 2922, 2855, 1717, 1375, 1332, 1152, 1021 cm ⁇ 1 .
- the 1 H and 13 C NMR spectra of phorbol ester II are shown in FIG. 7 and FIG. 8 , respectively.
- phorbol ester III is 12-O-(2E,4E)-6-oxohexa-2,4-dienoylphorbol-13-acetate with a molecular formula of C 28 H 34 O 9 deduced from HRESIMS m/z 537.20959 [M+Na] + (calcd for C 28 H 34 O 9 Na, 537.20950); [ ⁇ ] D 25 : +4.20 ⁇ 0.82 (c 0.1667, CHCl 3 ); UV (MeOH) ⁇ max (log ⁇ ): 295 (2.80), 249 (2.84) nm; and IR (neat) ⁇ max : 3413, 2925, 2855, 2360, 2339, 1625, 1597, 1261, 1184, 755 cm ⁇ 1 .
- the 1 H and 13 C NMR spectra of phorbol ester III are shown in FIG. 9 and FIG. 10 , respectively.
- phorbol ester IV is 12-O-(2E,4E)-6,7-dihydroxytetradeca-2,4-dienoylphorbol-13-acetate with a molecular formula of C 36 H 52 O 10 deduced from HRESIMS m/z 667.34515 [M+Na] + (calcd for C 36 H 52 O 10 Na, 667.34527); [ ⁇ ] D 25 : +10.44 ⁇ 1.45 (c 0.1667, CHCl 3 ); UV (MeOH) ⁇ max (log ⁇ ): 289 (2.79), 249 (2.83) nm; and IR (neat) ⁇ max : 3392, 2925, 2851, 1710, 1632, 1455, 1375, 1261, 1024, 802, 755 cm ⁇ 1 .
- the 1 H and 13 C NMR spectra of phorbol ester IV are shown in FIG. 11 and FIG. 12 , respectively.
- phorbol ester V is 12-deoxyphorbol 13-decanoate with a molecular formula of C 30 H 46 O 6 deduced from HRESIMS m/z 525.31921 [M+Na] + (calcd for C 30 H 46 O 6 Na, 525.31921); [ ⁇ ] D 25 : +8.55 ⁇ 0.63 (c 0.200, CHCl 3 ); UV (MeOH) ⁇ max (log ⁇ ): 325 (0.14), 249 (1.31) nm; and IR (neat) ⁇ max : 3392, 2925, 2356, 1710, 1629, 1335, 1155 cm ⁇ 1 .
- the 1 H and 13 C NMR spectra of phorbol ester V are shown in FIG. 13 and FIG. 14 , respectively.
- phorbol ester VI is 12-deoxyphorbol 13-octanoate with a molecular formula of C 28 H 42 O 6 deduced from ESIMS m/z 475 [M+H] + (calcd for C 28 H 43 O 6 ); [ ⁇ ] D 25 : +4.75 ⁇ 1.27 (c 0.200, CHCl 3 ); UV (MeOH) ⁇ max (log ⁇ ): 311 (0.01), 250 (2.85) nm; and IR (neat) ⁇ max : 3377, 2922, 2858, 1710, 1625, 1332, 1018 cm ⁇ 1 .
- the 1 H NMR spectrum of phorbol ester VI is shown in FIG. 15 .
- compositions of antiallergic phorbol ester and phorbol derivatives as the main active ingredients from the seeds of Aquilaria malaccensis were evaluated by the following assays.
- RBL-2H3 The mucosal mast cell-derived rat basophilic leukemia (RBL-2H3) cell line was purchased from the Bioresource Collection and Research Center (Hsin-Chu, Taiwan). Cells were grown in DMEM medium supplemented with 10% FBS and 100 U/mL penicillin plus 100 ⁇ g/mL streptomycin. Cells were cultured in 10 cm cell culture dishes at 37° C. in a humidified chamber with 5% CO 2 in air.
- compositions from the seeds of Aquilaria malaccensis were prepared for bioactive assays.
- the ethanolic extract EtOH
- n-butanol layer BuOH
- aqueous layer Water
- EtOAc ethyl acetate layer
- n-hexane layer Hexane
- 90% aqueous methanol layer MeOH
- the divided AM-4, AM4-4-7, AM4-4-8, AM4-4-9 were prepared for bioactive assays.
- a methylthiazol tetrazolium (MTT) assay was used to measure the potential toxic effects of the samples on RBL-2H3 cells (Chen et al., J. Nat. Prod., 72, 950-953, 2009). Briefly, RBL-2H3 cells (2 ⁇ 104 cells/well) were seeded in a 96-well plate overnight and treated with various concentrations of samples (10 ⁇ 100 ⁇ g/mL) for 24 h. MTT solution (0.5 mg/mL) was added to the wells (80 ⁇ L per well) and incubated for 1 h. The formed formazan crystals were dissolved in DMSO (80 ⁇ L).
- the absorbance at 595 nm was measured using microplate reader (Multiskan Ascent, Thermo Scientific). The degree of cell viability of each sample was calculated as the percentage of control value (untreated cells). The maximal tolerated dose of DMSO was 0.5%. All experiments were repeated at least two times.
- Degranulation ⁇ -hexosaminidase assay induced by A23187 or antigen The degree of A23187- and antigen-induced degranulation in RBL-2H3 cells was determined by a ⁇ -hexosaminidase release assay as described previously (Chen et al., J. Nat. Prod., 72, 950-953, 2009; Matsuda et al., Bioorg. Med. Chem., 12, 5891-5898, 2004) with following modifications.
- RBL-2H3 cells were seeded in a 96-well plate (2 ⁇ 10 4 cells/well) for A23187-induced and in 48-well plate (3 ⁇ 10 4 cells/well) for antigen-induced experiment.
- Cells were treated with various concentrations of the samples for 20 h.
- Dexamethasone (10 nM) was used as a positive control.
- the cells for the antigen-induced experiment were first sensitized with anti-DNP IgE (5 ⁇ g/mL) for at least 2 h.
- RBL-2H3 cells 4 ⁇ 10 4 cells/well
- Tyrode's buffer supplemented with 5.6 mM glucose, 2 mg/mL BSA and 2 mM glutamine was used to prepare the samples and treat the cells.
- 50 ⁇ L of supernatants were transferred into a 96-well microplate and examined as described above (the section of Cell viability assay).
- A23187 (1 ⁇ M) was used as a positive control. All experiments were repeated three times.
- Human neutrophils from venous blood of healthy, adult volunteers (20-30 years old) were isolated using a standard method of dextran sedimentation prior to centrifugation in a Ficoll-Hypaque gradient and hypotonic lysis of erythrocytes (Boyum et al., Scand. J. Clin. Lab. Invest., 97, 77-89, 1968).
- HBSS Hank's buffered salt solution
- Superoxide anion generation assay and elastase release inhibition assay Neutrophil superoxide anion generation was determined using superoxide dismutase (SOD)-inhibitory cytochrome reduction according to described procedures (Babior et al., J. Clin. Invest., 52, 741-744, 1973; Hwang et al., Free Radical Bio. Med., 41, 1433-1441, 2006). Degranulation of azurophilic granules was determined by measuring the elastase release as described previously (Hwang et al., Free Radical Bio. Med., 41, 1433-1441, 2006). All experiments were repeated at least three times.
- SOD superoxide dismutase
- MTT assay was used to according to the used in a previous manuscript. HepG2 (1 ⁇ 10 4 cells), A 549 (5 ⁇ 10 3 cells), and MDA-MB-231 (1 ⁇ 10 4 cells) were seeded into 96-well plates, followed by treatment with the AMS samples at concentration of 20 ⁇ g/mL After 72 h, the medium was removed and 100 ⁇ L of MTT solution (0.5 mg/mL) was added to each well. The plates were then incubated at 37° C. for 1 h and then, the MTT dye was detected by the addition of DMSO (100 ⁇ L). The absorbance was recorded at 550 nm. Doxorubicin was used as a positive control.
- antiallergic activity of the compositions from Aquilaria malaccensis seeds and A23187 was concluded in Table 1 whereas antiinflammatory effects of the compositions from A. malaccensis seeds on superoxide anion generation and elastase release in fMLP/CB-induced human neutrophils were summarized in Table 2.
- activity of phorbol ester-rich fraction (AM4) and phorbol ester I on stimulant-free degranulation in RBL-2H3 cells was shown in Table 4.
- the RBL-2H3 cells were treated with AM4 (10 ⁇ g/ml) and phorbol ester I (10 ⁇ g/ml) for 10 h.
- Tyrode's buffer supplemented with glucose, bovine serum albumin (BSA) and glutamine was used as a medium.
- A23187 (1 ⁇ M) was used as a positive control.
- the ethanolic extract (A-EtOH) showed potent antiallergic activity (IC 50 0.92 and 3.9 ⁇ g/mL in A23187 and antigen-induced ⁇ -hexosaminidase assay, respectively.
- antiallergic activity of the samples was due to inhibition of ⁇ -hexosaminidase release, and not false positive as a result of direct inhibition of ⁇ -hexosaminidase enzymatic activity (Wang et al., Biol. Pharm. Bull., 30, 388-392, 2007), the enzyme was extracted and tested with the active samples.
- AM4-4 (IC 50 4.8 ⁇ 10 +5 ⁇ g/mL, therapeutic index 1477328 , A 23187-induced; and IC 50 6.8 ⁇ 10 +4 ⁇ g/mL, therapeutic index 103776, antigen-induced (3-hexosaminidase assay) afforded the most active fraction AM4-4-8 (IC 50 7.6 ⁇ 10 ⁇ 6 ⁇ g/mL, therapeutic index 9645374, A23187-induced; and IC 50 8.0 ⁇ 10 +5 ⁇ g/mL, therapeutic index 9645374, antigen-induced degranulation assay), and a new compound, phorbol ester I (IC 50 values of 0.0017 ⁇ M, therapeutic index 71538, A23187-induced; and IC 50 0.011 ⁇ M, therapeutic index 10550, antigen-induced degranulation assay).
- the phorbol ester I really possesses the antiallergic activity and can be pharmaceutically applied for preventing and treating allergies.
- AM4-4-9 subfraction comprising phorbol ester I also really possesses the antiallergic activity and can be pharmaceutically applied for preventing and treating allergies. Therefore, AM4-3-13, AM4-4-3, and AM4-4-7 subfractions have the antiallergic activity correspondingly.
- the extractions and the fractionated fractions (phorbol ester-contained fractions) from Aquilaria malaccensis seeds can be pharmaceutically applied for preventing and treating allergies and show the potent antiallergic activity.
- this invention is characteristic for the advantages of easy collection, simple procedure, and plant growth no-effect due to the phorbol esters preparations from the seeds of Aquilaria malaccensis rather than the stems and barks. Besides, in accordance with the preparation methods of this invention, four new phorbol esters I ⁇ IV were isolated.
Abstract
Description
(e) Following bioactivity data, fraction AM4 (3212.0 g) was further fractionated over a Sephadex LH-20 column (CH2Cl2/MeOH, 1:1) to obtain eight sub-fractions (AM4-1 to AM4-8).
(f) Fraction AM4-3 (762.0 mg) was subjected to column chromatography (17 cm×4 cm,
(g) Fraction AM4-4 (173.7 mg) was further separated by column chromatography on silica gel (30 cm×1.5 cm, Geduran
(d) The MeOH layer was subjected to a column chromatography over silica gel (23 cm×4 cm,
(e) Fractions AM4 was further fractionated over a Sephadex LH-20 column (CH2Cl2/MeOH, 1:1) to obtain eight sub-fractions (AM4-1, 688.0 mg; AM4-2, 688.0 mg; AM4-3, 762.0 mg; AM4-4, 173.7 mg; AM4-5, 609.0 mg; AM4-6, 253.5 mg; AM4-7, 80.0 mg; and AM4-8, 80.0 mg). Of which subfractions, AM4-3 and AM4-4 are phorbol ester-rich fractions. The above scheme are shown in
(f) Fraction AM4-3 was subjected to column chromatography (17 cm×4 cm,
(g) Fraction AM4-4 was further separated by column chromatography on silica gel (30 cm×1.5 cm,
(h) From AM4-4-9, a new phorbol ester named 12-O-(2Z,4E,6E)-tetradeca-2,4,6-trienoylphorbol-13-acetate (43.9 mg) was isolated. This compound possesses a molecule of C36H50O8 and is represented by Formula I:
(i) From AM4-3-6 and AM4-4-3, a new phorbol ester named 12-deoxy-13-O-acetoylphorbol-20-octadec-9-enoate (8.8 mg) was afforded. This compound possesses a molecule of C40H62O7 and is represented by Formula II:
(j) From AM4-3-13, two new phorbol esters were given. One new phorbol ester named 12-O-(2E,4E)-6-oxohexa-2,4-dienoylphorbol-13-acetate (0.7 mg) with a molecule of C28H34O9 is represented by Formula III. Another new phorbol ester named 12-O-(2E,4E)-6,7-dihydroxytetradeca-2,4-dienoylphorbol-13-acetate (0.9 mg) possesses a molecule of C36H52O10 and is represented by Formula IV.
(k) From AM4-4-7 and AM4-4-8, two known phorbol esters, 12-deoxyphorbol 13-decanoate (8.5 mg) and 12-deoxyphorbol 13-octanoate (1.4 mg), were isolated. These two compounds possess molecules H of C30H46O6 and C28H42O6 and are represented by Formulas V and VI, respectively.
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